Aquaporin 4 (AQP4) is a water specific member of the Aquaporin family of water and water/glycerol transporters (Hasegawa, et al. J. Biol. Chem. 1994, 269, 5497). The aquaporins are a structurally unique class of transmembrane transporter proteins, particularly compared to other transporters such as the sodium, calcium and chloride channels. Unlike the typical channel proteins, where the active channel is formed at the nexus of four or more protein monomers, the aquaporins are characterized by the formation of a protein homotetramer, where each protein monomer contains a channel that is virtually independent from that of the other protein monomers (Hiroaki, et al. J. Mol. Bio. 2006, 355, 628). The nexus of the AQP protein monomers is not believed to form an active channel or pore. Moreover, the significance of the aquaporin channel structure, versus that of other transmembrane channels, is not understood.
The AQP4 water transporter is widely distributed in the human body, with particularly high concentrations in the brain, eyes, ears, muscles, lungs and kidneys (Jung, et al. Proc. Nat. Acad. Sci., USA 1994, 91, 13052-13056). It is highly conserved among mammalian species, typically showing greater than 95% identity, and also has a similar biological distribution (Zardoya, et al. J. Mol. Evol. 2001, 52, 391). The main role of AQP4 is generally regarded to be the regulatory water balance of the tissues it is localized in.
Since the initial discovery of AQP4, chemical modulators thereof have been considered potential therapeutic agents for a variety of pathologies (Castle. DDT2005, 10, 485; Kobayashi, et al. J. Pharmocol. Sci. 2004, 96, 264; Wang, et al. Acta Pharmacol. Sin. 2006, 27, 395). However, the relationship between AQP4 and disease may not be completely obvious, and in many cases may not reflect a pathological change in AQP4 expression or regulation. Indeed AQP4 plays a critical role in cellular migration and the cytoskeletal morphology of the tissues it is expressed in, and is also highly responsive to changes in the ionic strength of both the cytosol and extracellular fluid, and the function of the various transporters and enzymes that control that ionic strength (Simard, et al. Neuroscience, 2004, 129, 877). The use of pharmaceutical agents to modulate AQP4 function as a means of treating human disease has gained more relevance considering the recent understanding of how that function can be related to inflammation, in general (Ito, et al. J. Neurochem. 2006, 99, 107) and neuroresponse in particular (Tian, et al. Nat. Med. 2005, 11, 973; Binder, et al. Glia 2006, 54, 358).
The use of AQP4 modulators for the treatment of cerebral edema has been considered the most promising match for this target (Papadopoluos, et al. Pediatr Nephrol. 2007, in press). Currently the use of AQP4 inhibitors for the treatment of cytotoxic edema appears to be well supported (Amiry-Moghaddam, et al. FASEB J. 2004, 18, 542; Aoki-Yoshino, et al. Acta Neuropathol. 2005, 110, 281; Manley, et al. Nat. Med. 2000, 6, 159), while the treatment of vasogenic edema now appears to be contravened. The edema related conditions of ischemia (Xiao. Acad. Emerg. Med. 2002, 9, 933), brain tumors (Saadoun, et al. J. Clin. Pathol. 2003, 56, 972); Warth, et al. Acta Neuropathol. 2005, 109, 418), eclampsia (Quick, et al. FASEB J. 2005, 19, 170), meningitis (Papadopoulos, et al. J. Biol. Chem. 2005, 280, 13906), Creutzfeldt-Jakob disease (Rodriguez, et al. Acta Neuropathol. 2006, 112, 573), and lupus cerebritis (Alexander, et al. Biochim. Biophys. Acta 2003, 169) appear to be particularly well suited for pharmacological treatment by AQP4 modulators, based on the evidence of the biophysiological studies reported therein.
AQP4 modulating compounds have been suggested as medicants for the treatment of neurological disorders, particularly epilepsy (Binder, et al. Neuroreport 2004, 15, 259; Binder, et al. Glia 2006, 54, 358), human immunodeficiency virus related dementia (St Hillaire, et al. J. Neurovirol. 2005, 11, 535), neuromyelitis optica (Lennon, et al. J. Exp. Med. 2005, 202, 473) and drug addiction (Li, et al. Neurosci. Lett. 2006, 403, 294) based on the specifics of the disease pathology, results from biophysical studies or a combination thereof. More recent studies have shown the presence of significant AQP4 expressional upregulation in the early stages of Alzheimer disease (Pérez, et al. Brain Res. 2007, 1128, 164).
In addition to the cerebral and neurological disorders, a number of peripheral diseases have been found to share a relationship with AQP4. Ocular diseases relating to retinal ischemia (Bringmann, et al. Acta Ophthalmol. Scand. 2005, 83, 528), glaucoma (Verkman. Exp. Eye Res. 2003, 76, 137), proliferative retinopathy (Tenckhoff, et al. Neuroreport 2005, 16, 53) and bullous keratopathy (Kenney, et al. J. Histochem. Cytochem. 2004, 52, 1341) also appear to be related to AQP4 function, and hence can be considered as being in part treatable by direct AQP4 modulating agents. Muscular dystrophy is known to be directly related to problems associated with AQP4 localization and changes in its expression level (Compton, et al. J. Neuropathol. Exp. Neurol 2005, 64, 350, Jimi, et al. Pathol. Res. Prac. 2004, 200, 203); moreover, some classes of tumors overexpress AQP4 as part of their water recruitment process (Carmosino, et al. Biol. Cell 2005, 97, 735), both of which are considered potential therapeutic areas for AQP4 modulating agents.
To date, no pharmacologically relevant compounds have been demonstrated that are capable of directly inhibiting AQP4 in any assay system (either in vitro or in vivo). Recently, a patent application claiming a series of quaternary ammonium compounds as aquaporin inhibitors has been published (Deen, et al. PCT WO 2005/094806 A1, 2005). However, of the exemplary compounds, only tetraethyl ammonium chloride was shown to inhibit AQP4, and it appeared that compounds from that general class could not be considered as AQP4 inhibitors in any general sense. A similar result was also demonstrated in a subsequent paper (Detmers, et al. J. Biol. Chem. 2006, 281, 14207). Moreover, an earlier patent application has claimed the use of AQP inhibiting agents as therapeutic compounds for lowering interocular pressure, in the treatment of ophthalmologic diseases (Wax, PCT WO 2004/069181 A2, 2004). Again, no evidence was demonstrated regarding the particular types of compounds that would be able to inhibit AQP4, either specifically or as a pan-inhibitor of AQP isozymes. Nocodazole, in particular, was described as inhibiting the AQP4 mediated hypoosmotic expansion of HEK293 cells (van den Kieboom, et al. PCT WO 01/64219 A2, 2001); however, the HEK293 cell line expresses AQP4 amongst other AQP isozymes, and no data was shown to demonstrate which isozyme that molecule was interacting with. It is particularly relevant because nocodazole is a known inhibitor of AQP2 (Tajika, et al. Histochem. Cell. Biol. 2005, 124, 1), which is also expressed by the HEK293 cell line.
In addition to the compounds described above in the context of AQP4 inhibition, a number of modulators for other AQP isozymes were reported. Phloretin had had been described as being a pan inhibitor of several AQP subtypes (Tsukaguchi, et al. J. Am. Physiol. Soc. 1999, 277, F685), most notably AQP9, but there was no particular indication that this compound could be used to modulate the water transport of AQP4. AQP1 was shown to be inhibited in a dose dependent manner by the pan carbonic anhydrase inhibitor acetazolamide (Ma, et al., Acta Pharmacol. Sin. 2004, 25, 90). Many of the AQP transporter subtypes have also been shown to be inhibited by heavy metal salts (Kuwahara, et al., Biochemistry 1997, 36, 13973), presumably because of binding to an intra-pore cystine residue. However, AQP4 lacks such a residue in its pore region, and has also been shown to be insensitive to heavy metal inhibition (J Jung, et al. Proc. Nat, Acad. Sci., USA 1994, 91, 13052).
Hence at the time of this invention, pharmacologically relevant modulators of AQP4 were widely believed to be valuable as therapeutic agents; however, no potential agents had been identified.